The mammalian ovarian follicle consists of a multilayered complex of somatic cells that surrounds the oocyte. A signal from the follicle cells keeps the oocyte cell cycle arrested at prophase of meiosis I until luteinizing hormone from the pituitary acts on the follicle cells to release the arrest, causing meiosis to continue. Maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on an elevated level of cAMP in the oocyte. To investigate how the cAMP level is regulated, we examined whether the activity of an oocyte G-protein of the family that stimulates adenylyl cyclase (Gs) is required to maintain meiotic arrest. Microinjection of a dominant negative form of Gs into Xenopus and mouse oocytes, or microinjection of an antibody that inhibits the Gs G-protein into zebrafish oocytes, caused meiosis to resume. Together with previous studies, these results support the conclusion that Gs-regulated generation of cAMP by the oocyte is a common mechanism for maintaining meiotic prophase arrest in vertebrate oocytes.